1. Field of the Invention
This invention relates to use of a method for screening nonpathogenic anti-inflammatory bacterial strains, and products and methods using such strains for treatment and prophylaxis of unwanted inflammation caused by certain bacteria or other inflammation-causing agents.
2. Description of the Related Art
Monocytes leave the bone marrow and travel through the peripheral blood vessels until they reach the mucosa/serosa of the gastro intestinal tract. These putative macrophages are key to the interaction and propagation of the signals necessary to regulate the immune system of the GI.
In the gastrointestinal tract, there is a constant level of immune response in the macrophages of the mucosal epithelium to the bacteria in the intestinal lumen and attached to the intestinal mucosa. In the normal state, this response involves the generation of cytokine signals to restrict and contain an unnecessary inflammatory response. However, when a pathogen or toxin is presented to these cells, they form the first line of defense and react by producing an increasing amount of pro-inflammatory cytokines, which propagate the inflammatory response until the threat is removed. The generation of cytokines relevant to the interactions with commensal (non-threatening) bacteria as well as those involved in the full inflammatory response to pathogens, are subject to intervention by lactic acid bacteria themselves (including surface antigens) or by substances produced by these lactic acid bacteria and it is clear that the commensal flora has extensive interaction with the macrophages of the mucosa to maintain a balanced reaction to the gut flora and thereby maintain optimal health (Rook G A, Adams V, Hunt J, Palmer R, Martinelli R, Brunet LR. Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders. Springer Semin Immunopathol 2004; 25:237-255).
It is known that various pathogens can cause inflammation, for example in the gastrointestinal tract. Such inflammation, for example, in the stomach and gastrointestinal tract, is mediated by intercellular signal proteins known as cytokines which are produced by macrophages and dendritic cells in the epithelium in response to an antigenic stimulus such as that produced by a pathogen. Upon contact between the epithelium and the antigen of a pathogen or endotoxins produced by it, such as LPS, antigen presenting cells (including dendritic cells) in the epithelium propagate the signal to naive macrophages which then respond in a so-called Th-1 type response where pro-inflammatory cytokines including TNFα, IL-1, IL-6, IL-12 are produced by the macrophages. These cytokines in turn stimulate natural killer cells, T-cells and other cells to produce interferon γ (IFNγ), which is the key mediator of inflammation. IFNγ leads to an escalation of the inflammatory response and the reactions described above that lead to cytotoxicity. Naive macrophages can also respond to antigens with a Th-2 type response. This response is suppressed by IFNγ. These Th-2 type cells produce anti-inflammatory cytokines such as IL-4, IL-5, IL-9 and IL-10.
IL-10 is known to inhibit the production of IFNγ and thus dampen the immune response. The balance between Th-1 and Th-2 type cells and their respective cytokine production defines the extent of the inflammation response to a given antigen. Th-2 type cells can also stimulate the production of immunoglobulins via the immune system. Anti-inflammatory activity in the gastrointestinal tract, where there is a reduced TNFα level, correlates with enhanced epithelial cells (gut wall lining integrity) and thus to a reduction in the negative effects caused by gastrointestinal pathogens and toxins.
The results of a number of research studies indicate that DNA can exert an anti-inflammatory action on intestinal epithelial cells, or can stimulate the immune system (Madsen et al. and Rachmilewitz et al, respectively, presentations at Digestive Disease Week, May 19-22, 2002, The Moscone Center, San Francisco).
Inflammation is involved in several diseases in mammals both externally on skin, eyes, etc., and internally for example on various mucous membranes; in the mouth, GI tract, vagina etc. but also in muscles, bone-joints and in brain-tissue. In the GI-tract there are several diseases connected to inflammation, for example, gastritis, ulcer and inflammatory bowel disease (IBD). IBD is a chronic disorder that causes an inflamed and swollen digestive tract or intestinal wall. When the digestive tract becomes inflamed or swollen with IBD, sores (ulcers) form and bleed. This in turn can cause abdominal pain, watery diarrhea, blood in the stool, fatigue, reduced appetite, weight loss, or fever. Thus, inflammation in IBD leads to tissue damage such as ulcers and exacerbated disease in a patient.
The two most common forms of IBD are ulcerative colitis (UC) and Crohn's disease (CD). Crohn's Disease is a chronic condition characterised by recurrent inflammatory lesions of the entire gut wall from mucosa to serosa and can affect many sites in the intestine. The disease has been linked to imbalances in the gut microflora and an overexpressed inflammatory reaction to components of the normal gut flora and this reaction is currently treated with poor success using a series of different drugs, one of which is based on anti-TNFα therapy designed to reduce the levels of TNFα in the gastro-intestinal mucosa. Thus, persons with such a disease form an ideal study group and indeed target group for the use of immuno-modulatory, inflammation attenuating lactobacilli. 
Mice spontaneously develop chronic colitis, which does not occur in germ-free animals. Mouse colitis is similar to human Crohn's disease, a chronic serious inflammatory disease of the gastrointestinal tract. Crohn's disease usually occurs in the intestines, but may occur anywhere in the gastrointestinal tract. These conditions require the presence of enteric bacteria and are both Th1-mediated-IL-12-dependent forms of colitis.
Because of the similarities of the causes and symptoms, mouse models of colitis and other mouse models are often used to study components of the inflammatory response directly, and, as the same mechanisms are assumed to apply in man, are often accepted to be used as models to develop treatments for human gastrointestinal disease. There are, however, some questions about the relevance of the animal derived models for humans, so there is a need to have alternative methods to study the human mechanisms and to confirm results from other models, in more human based systems. The purpose of the invention herein is to provide a method based on human cells to select lactic acid bacteria that display anti-inflammatory characteristics and then to use such selected lactic acid bacteria for prophylaxis and treatment of various inflammatory diseases.
Lactobacillus reuteri is one of the naturally occurring inhabitants of the gastrointestinal tract of animals and is routinely found in the intestines of healthy animals and despite the low pH, occasionally also in the human stomach. It is known to have antibacterial activity. See, for example U.S. Pat. Nos. 5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. When L. reuteri cells are grown under anaerobic conditions in the presence of glycerol, they produce the antimicrobial substance known as reuterin (β-hydroxy-propionaldehyde). It is also known that certain strains of lactobacilli including L. reuteri have anti-inflammatory properties as is in U.S. patent application 20040208863 and 20040067573. Other immunomodulating activity has also been associated with various other lactobacilli. 
Human clinical trials and animal studies have shown that Lactobacillus can prevent or improve inflammation in chronic colitis. It is hypothesised that lactobacilli are capable of down-regulating pro-inflammatory cytokine responses induced by enteric bacteria. J. Peña et al (2003) investigated whether lactobacilli diminish production of tumour necrosis factor alpha (TNF α) by a murine macrophage line. It was shown that TNF-α production by murine macrophages incubated with Lactobacillus rhamnosus GG and lipopolysaccharides was significantly inhibited compared to controls.
The same group of workers investigated whether Lactobacillus spp. might be effective in ameliorating Helicobacter-induced inflammation in vivo. For this, co-inoculation experiments with L. reuteri and L. paracasei were performed in the H. hepaticus mouse colitis model. A significant decrease in pathology scores was observed in female mice that have received both H. hepaticus and Lactobacillus spp. when compared to animals given H. hepaticus alone. The probiotic effect of Lactobacillus spp. in this model was shown to be independent of the exclusion of H. hepaticus bacteria, but dependent on post-transcriptional regulation of TNF-α expression. The data suggests the existence of a novel mechanism by which Lactobacillus spp. can down-regulate Helicobacter induced pro-inflammatory cytokine production in macrophages.
Patent WO2004/031368A1 describes Lactobacillus strains selected for its ability to reduce gastrointestinal inflammation associated with H. pylori infection in mammals using a mouse macrophage assay for TNF-α activity.
Patent application US20040057943A1 relates to a process for selection of new probiotic strain of L. coryniformis, L. salivarius, L. acidofilus, L. gasseri and L. fermentum that are capable of surviving in breast milk and/or amniotic fluid.
S. Ménard et al. (2003) investigated whether lactic acid bacteria secreted metabolites that retain anti-inflammatory properties after intestinal transport. Analysis of LPS binding to THP-1 pro-monocytes in the presence of bacteria conditioned media (CM) was made to see if LPS has to bind to LPS binding protein (LBP) before LPS-LBP can recognize the CD14 receptor. The binding was measured by flow cytometry. It was shown that Bifidobacterium and Streptococcus thermofilus CM completely inhibited LDB dependent LPS binding to THP-1 cells by releasing metabolites exerting an anti TNF-α effect capable of crossing the intestinal barrier.
While the differences in the ability of several lactobacilli to reduce inflammation, including gastrointestinal inflammation, is known, it was not previously known that these differences are better predicted by using a model based on human cells and that such a model is preferred to select such strains for potential effect in humans.
It is therefore an object of the invention to provide strains of lactic acid bacteria, which have been selected using human cell lines, for their capability of reducing inflammation, such as that due to IBD. It is a further object of the invention to provide products containing said strains, including agents for treatment or prophylaxis of inflammation, for example associated with IBD for administration to humans and other mammals, including conditioned media in which said strains have grown and protein-containing extracts thereof. Thus, the invention is used to treat inflammation in IBD which leads to tissue damage such as ulcers and exacerbated disease in a patient.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.